Examination of Targeted Mutagenesis Effects via Quick Change at Leucine ۲۶۲ and Its Conversion to Arginine on Uricase Enzyme Structure

Efforts to enhance the stability of uricase under various conditions, particularly temperature, have garnered significant attention from researchers. Targeted mutagenesis is a technique employed to stabilize proteins such as the uricase enzyme. This enzyme belongs to the oxidoreductase class and plays a crucial role in the oxidation of uric acid as substrate and its conversion to allantoin, a process associated with reduced levels of uric acid, thereby preventing diseases such as gout and hyperuricemia. In this study, after analyzing and selecting the mutation site at position Leucine ۲۶۲ based on existing interactions, a mutation to Arginine was introduced, and the corresponding forward and reverse primer was designed and synthesized by sinaclone company. Following the PCR reaction and product transfer to competent cells by heat shock method, plasmid extraction was performed, and the accuracy of the mutation was verified through sequencing. Cultivation of bacteria from BL۲۱ strain, expression by IPTG, and purification of the mutated uricase were conducted, and its activity was measured and compared with that of the wild-type enzyme.

کلیدواژه ها:

Uricase ، targeted mutagenesis ، Quick change ، Leucine ۲۶۲ ، PCR reaction